A 35-year hunt to uncover a lost work by Leonardo Da Vinci is reaching a climax in Florence, while also facing an angry protest by more than 100 prominent art historians who deplore the destructive but speculative work possibly leading to the masterpiece's uncovery.

The row centres on a wall in Florence's Palazzo Vecchio on which is painted a 16th century fresco. But, according to researcher Maurizio Seracini, this wall conceals another wall on which Da Vinci bgan painting The Battle of Anghiari, a monumental battle scene that is considered by some his finest work.

Seracini, who works at the University of California, San Diego, and is featured in Dan Brown's mystery The Da Vinci Code, inserted tiny cameras through drilled holes in the visible wall a week ago, and found a 2cm cavity. On the back wall beyond the cavity, traces of an organic pigment were found, convincing some that the Da Vinci masterpiece exists. With full results expected in the new year, the city'ss mayor, Matteo Renzi claimed: "We are finally there – after five centuries we are able finally to resolve this mystery."

But 150 art historians from museums including the New York Met and the National Gallery in London have signed a petition to stop the work, angry at the fact that holes are being drilled in the front wall bearing its own renown fresco, Giorgio Vasari's The Battle of Marciano in Val di Chiana, painted in 1563.

"We also believe that Da Vinci painted on the wall opposite, but Seracini just doesn't know his art history," said Tomaso Montanari, the art history professor who started the petition. Backing the experts, the heritage group Italia Nostra has complained to Florence magistrates, who have opened an investigation.

"This is a wasted expense when we need every penny for restoring the art we have," said Italia Nostra president Alessandra Mottola Molfino. "Instead of restoring the Vasari fresco we are drilling holes in it."

Da Vinci started in 1504 on his battle scene using an experimental oil paint technique which failed miserably, dripping before it dried and leading him to abandon the work. Those scenes he completed were however widely copied, including by Rubens, whose drawing of one scene hangs in the Louvre. After 1555 the room was renovated, and Da Vinci's half-finished painting was lost.

Seracini's suspicion is that Vasari was loth to destroy Da Vinci's work, and preferred to brick it up behind a new wall and add his fresco on this. This hunch was stimulated when he found that in his fresco Vasari painted a soldier holding a flag on which is written: "He who seeks, finds." Using radar, Seracini then found the cavity behind the fresco.

But Montanari is not convinced. "Vasari knew how to remove works by other people while keeping them intact. What sense would there have been sealing up the Da Vinci, unless you get into childish Dan Brown logic?"

Montanari bagan the petition last week when Cecilia Frosinone, an expert with a Florence art restoration institute working with Seracini, resigned on "ethical" grounds after the culture minister gave permission to drill seven holes in the Vasari fresco. "We don't have external controls on the work any more, and that is what we want restored," said Montanari.

On Monday Seracini described the petition as sour grapes, an attempt "by the excluded to block extraordinary research", adding: "This demagogic attack risks Italy being derided around the world."